The ABCA4 gene is the most frequently mutated Mendelian retinopathy-associated gene. Biallelic variants lead to a variety of phenotypes, however, for thousands of cases the underlying variants remain unknown. Here, we aim to shed further light on the missing heritability of ABCA4-associated retinopathy by analyzing a large cohort of macular dystrophy probands. A total of 858 probands were collected from 26 centers, of whom 722 carried no or one pathogenic ABCA4 variant, while 136 cases carried two ABCA4 alleles, one of which was a frequent mild variant, suggesting that deep-intronic variants (DIVs) or other cis-modifiers might have been missed. After single molecule molecular inversion probes (smMIPs)-based sequencing of the complete 128-kb ABCA4 locus, the effect of putative splice variants was assessed in vitro by midigene splice assays in HEK293T cells. The breakpoints of copy number variants (CNVs) were determined by junction PCR and Sanger sequencing. ABCA4 sequence analysis solved 207 of 520 (39.8%) naive or unsolved cases and 70 of 202 (34.7%) monoallelic cases, while additional causal variants were identified in 54 of 136 (39.7%) probands carrying two variants. Seven novel DIVs and six novel non-canonical splice site variants were detected in a total of 35 alleles and characterized, including the c.6283-321C>G variant leading to a complex splicing defect. Additionally, four novel CNVs were identified and characterized in five alleles. These results confirm that smMIPs-based sequencing of the complete ABCA4 gene provides a cost-effective method to genetically solve retinopathy cases and that several rare structural and splice altering defects remain undiscovered in Stargardt disease cases.

• MeSH
• ABC transportéry genetika   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• makulární degenerace * genetika   MeSH
• mutace genetika   MeSH
• retinální dystrofie * genetika   MeSH
• sekvenční analýza MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Importance: The mechanisms behind the phenotypic variability and reduced penetrance in autosomal recessive Stargardt disease (STGD1), often a blinding disease, are poorly understood. Identification of the unknown disease modifiers can improve patient and family counseling and provide valuable information for disease management. Objective: To assess the association of incompletely penetrant ABCA4 alleles with sex in STGD1. Design, Setting, and Participants: Genetic data for this cross-sectional study were obtained from 2 multicenter genetic studies of 1162 patients with clinically suspected STGD1. Unrelated patients with genetically confirmed STGD1 were selected. The data were collected from June 2016 to June 2019, and post hoc analysis was performed between July 2019 and January 2020. Main Outcomes and Measures: Penetrance of reported mild ABCA4 variants was calculated by comparing the allele frequencies in the general population (obtained from the Genome Aggregation Database) with the genotyping data in the patient population (obtained from the ABCA4 Leiden Open Variation Database). The sex ratio among patients with and patients without an ABCA4 allele with incomplete penetrance was assessed. Results: A total of 550 patients were included in the study, among which the mean (SD) age was 45.7 (18.0) years and most patients were women (311 [57%]). Five of the 5 mild ABCA4 alleles, including c.5603A>T and c.5882G>A, were calculated to have incomplete penetrance. The women to men ratio in the subgroup carrying c.5603A>T was 1.7 to 1; the proportion of women in this group was higher compared with the subgroup not carrying a mild allele (difference, 13%; 95% CI, 3%-23%; P = .02). The women to men ratio in the c.5882G>A subgroup was 2.1 to 1, and the women were overrepresented compared with the group carrying no mild allele (difference, 18%; 95% CI, 6%-30%; P = .005). Conclusions and Relevance: This study found an imbalance in observed sex ratio among patients harboring a mild ABCA4 allele, which concerns approximately 25% of all patients with STGD1, suggesting that STGD1 should be considered a polygenic or multifactorial disease rather than a disease caused by ABCA4 gene mutations alone. The findings suggest that sex should be considered as a potential disease-modifying variable in both basic research and clinical trials on STGD1.

• MeSH
• ABC transportéry genetika   metabolismus   MeSH
• alely MeSH
• DNA genetika   MeSH
• frekvence genu MeSH
• genotyp MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace * MeSH
• mutační analýza DNA MeSH
• průřezové studie MeSH
• rozložení podle pohlaví MeSH
• sexuální faktory MeSH
• Stargardtova nemoc diagnóza   genetika   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH

PURPOSE: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. METHODS: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. RESULTS: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. CONCLUSION: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

• MeSH
• ABC transportéry genetika   MeSH
• genomika MeSH
• introny MeSH
• lidé MeSH
• makulární degenerace * genetika   MeSH
• mutace MeSH
• rodokmen MeSH
• Stargardtova nemoc MeSH
• transkriptom * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Stargardt disease (STGD) leads to blindness in children and young adults. So far, no curative therapy is available and gene augmentation therapies have not yet advanced to the clinics, in part, due to the limited packaging capacity of adeno-associated viruses used to transfer genes into photoreceptor cells. Prime editing offers a new perspective to treat mutations on the genomic level. A nicking variant of Cas9 fused to a reverse transcriptase complex with an elongated guideRNA force intracellular mismatch repair to correct the targeted mutation even in postmitotic cells such as photoreceptors in the eye. Using a custom-made bioluminescence resonance energy transfer (BRET)-based editing sensor in HEK293 cells, we tested 27 different prime editing guide RNAs (pegRNAs) and additional 4 nicking guide RNAs (ngRNAs) with regard to their efficiency to induce sequences changes in exon 43 of the porcine ATP binding cassette subfamily A member 4 (ABCA4) gene that eliminate a mutagenic adenine frameshift insertion, which has been associated with STGD in humans. We identified nine working pegRNAs, and in combination with ngRNAs, we achieved a correction rate of up to ≈92% measured with the BRET-based reporter system. Our data prove the high efficiency of prime editors to correct mutations and highlight the importance of optimal ngRNA design, thus offering a promising editing tool to correct ABCA4 mutations in the disease context.

• MeSH
• ABC transportéry * genetika   MeSH
• dítě MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mladý dospělý MeSH
• mutace MeSH
• prasata MeSH
• přenos energie MeSH
• Stargardtova nemoc genetika   MeSH
• zvířata MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladý dospělý MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cíl: Popsat klinický nález a provést molekulárně genetickou analýzu u dvou probandů českého původu s diagnózou Stargardtovy choroby. Poskytnout souhrn praktických poznatků plynoucích z mutační analýzy genu ABCA4 a poukázat na některé problematické aspekty spojené se screeningem tohoto genu. Metody: U obou mužů ve věku 39 a 26 let jsme provedli oční vyšetření včetně optické koherenční tomografie se spektrální doménou (SD-OCT). DNA byla izolována z venózní krve. Identifikace patogenních sekvenčních variant byla provedena pomocí genotypovacího mikročipu pro gen ABCA4, verze 11.0 (Asper Ophthalmics, Estonsko) detekující 558 známých mutací a jednonukleotidových polymorfismů. Výsledky: U prvního probanda byla nejlepší korigovaná zraková ostrost obou očí 0,1, u druhého probanda 0,05. Klinické vyšetření prokázalo typický nález atrofie makuly s přítomností žlutavých skvrn na očním pozadí. Vyšetření SD-OCT odhalilo oboustranně chybění linie elipsoidů vnitřních segmentů fotoreceptorů, také v literatuře označovaná jako linie junkce vnitřních a zevních segmentů fotoreceptorů, ztenčení neuroretiny a nepravidelnosti pigmentového epitelu sítnice. V souladu s autozomálně recesivním typem přenosu byla rodinná anamnéza pro Stargardtovu chorobu v obou případech negativní. Molekulárně genetickým vyšetřením bylo zjištěno, že první proband je nositelem mutace c.4234C>T; p.(Gln1412*) v exonu 28 a c.5882G>A; p.(Gly1961Glu) v exonu 42. U druhého probanda byla detekována pouze jedna známá patogenní mutace c.1988G>A p.(Trp663*) v exonu 14. Závěr: Poprvé byly u českých pacientů zjištěny mutace zodpovědné za vznik Stargardtovy choroby. Nemožnost nalézt jednu nebo obě patogenní sekvenční varianty je u této choroby za použití současných metod poměrně častým jevem. Budoucí studie by se měly zaměřit na stanovení spektra a frekvence jednotlivých mutací v ABCA4 u rozsáhlejšího souboru pacientů českého původu. Znalost příčinných mutací zlepšuje klinické poradenství pacientům a v indikovaných případech umožňuje preimplantační diagnostiku.

Purpose: The aim of our study was to describe the phenotype and to perform molecular genetic investigation in two probands of Czech origin diagnosed with Stargardt disease (STGD). Methods: Both males underwent ocular examination including assessment by high-resolution spectral domain optical coherence tomography (SD-OCT). DNA was isolated from venous blood. Mutation detection was performed using the ABCA4 genotyping microarray (Asper Ophthalmics, Estonia). Results: The best corrected visual acuity in proband 1 (aged 39 years) was 0.1 bilaterally, and 0.05 in proband 2 (aged 26 years). Fundus examination showed typical multiple yellow-white lesions and macular atrophy. Alterations of retinal pigment epithelium, retinal thinning and disruption of the photoreceptor inner segment ellipsoid band were detected with an SD-OCT. Two known disease-causing mutations in ABCA4 were identified in proband 1; c.4234C>T, p.(Gln1412*) in exon 28; and c.5882G>A, p.(Gly1961Glu) in exon 42. Only one pathogenic change was detected in proband 2; c.1988G>A, p.(Trp663*) in exon 14. A second change, anticipated because of the recessive status of the disease, was not identified. Conclusion: The frequency and full spectrum of ABCA4 mutations in Czech patients with inherited retinal disorders is yet to be established. The inability to detect a second pathogenic change in ABCA4 coding sequences in proband 2 warrants further investigation.

• Klíčová slova
• Stargardtova choroba, SD-OCT, ABCA4,
• MeSH
• ABC transportéry * genetika   MeSH
• degenerace retiny diagnóza   genetika   patologie   MeSH
• dospělí MeSH
• exony genetika   MeSH
• genotypizační techniky MeSH
• lidé MeSH
• makulární degenerace diagnóza   MeSH
• mutace genetika   MeSH
• mutační analýza DNA * MeSH
• nemoci retiny * diagnóza   genetika   MeSH
• optická koherentní tomografie MeSH
• pigmentový epitel oční patologie   MeSH
• poruchy zraku diagnóza   genetika   patofyziologie   MeSH
• vnitřní segment fotoreceptoru sítnice patologie   MeSH
• zraková ostrost MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH

Cíl: Cílem práce je poskytnout informace o probíhajících klinických studiích testujících účinnost a bezpečnost nových terapeutických postupů u Stargardtovy choroby. Metody: Po zadání hesla „Stargardt“ byl prohledán mezinárodní registr klinických studií (www.clinicaltrials.gov) a vytvořen seznam aktivních studií. Výsledky: V současné době je registrováno osm klinických studií, které pacientům se Stargardtovou chorobou nabízejí možnost zařazení. Všechny studie jsou ve fázi I nebo II a podle mechanismu účinku zkoušeného přípravku je lze rozdělit do čtyř skupin: zabránění vzniku toxických dimerů vitaminu A, genová terapie obnovující funkčnost genu ABCA4, ochrana buněk sítnice před oxidativním poškozením a náhrada poškozeného pigmentového epitelu sítnice a fotoreceptorů pomocí kmenových buněk. Základní podmínkou pro zařazení do většiny těchto studií je potvrzení klinické diagnózy na molekulárně genetické úrovni, tedy znalost příčinných mutací. Závěr: Registrované klinické studie testující nové metody léčby Stargardtovy choroby dávají naději, že průběh této choroby bude možno již v blízké budoucnosti aktivně ovlivnit.

Purpose: To provide information on currently ongoing clinical trials for Stargardt disease. Methods: We have searched the clinical trial register (www.clinicaltrials.gov) for the keyword „Stargardt“ and list active ongoing studies. Results: There are currently eight registered clinical trials enrolling patients with Stargardt disease; all in phase I or II aiming at four mechanisms of action: inhibition of the production of vitamin A toxic dimers, gene therapy restoring wild type transcription of the ABCA4 gene, neuroprotection preventing retinal cells from oxidative damage, and replacement of the damaged retinal pigment epithelium using stem cell therapy. The basic prerequisite for enrolment in the vast majority of clinical trials is confirmation of the clinical diagnosis by mutational analysis. Conclusion: The wide variety of therapies that are registered as clinical trials for Stargardt disease significantly raises the possibility that effective treatments will be available in the near future for this currently incurable condition and that molecular genetic testing should be increasingly considered.

• Klíčová slova
• Stargardtova choroba, ABCA4,
• MeSH
• ABC transportéry genetika   MeSH
• buněčná a tkáňová terapie MeSH
• Crocus MeSH
• dědičné nemoci očí MeSH
• degenerace retiny * terapie   vrozené   MeSH
• fotoreceptory obratlovců patologie   MeSH
• fytoterapie * MeSH
• genetická terapie * metody   MeSH
• klinické zkoušky, fáze I jako téma MeSH
• klinické zkoušky, fáze II jako téma MeSH
• lidé MeSH
• mutace MeSH
• pigmentový epitel oční patologie   MeSH
• vitamin A terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

• Klíčová slova
• Stargardtova choroba, SD-OCT, ABCA4,
• MeSH
• ABC transportéry * genetika   MeSH
• degenerace retiny diagnóza   genetika   patologie   MeSH
• dospělí MeSH
• exony genetika   MeSH
• genotypizační techniky MeSH
• lidé MeSH
• makulární degenerace diagnóza   MeSH
• mutace genetika   MeSH
• mutační analýza DNA * MeSH
• nemoci retiny * diagnóza   genetika   MeSH
• optická koherentní tomografie MeSH
• pigmentový epitel oční patologie   MeSH
• poruchy zraku diagnóza   genetika   patofyziologie   MeSH
• vnitřní segment fotoreceptoru sítnice patologie   MeSH
• zraková ostrost MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH

• Klíčová slova
• Stargardtova choroba, ABCA4,
• MeSH
• ABC transportéry genetika   MeSH
• buněčná a tkáňová terapie MeSH
• Crocus MeSH
• dědičné nemoci očí MeSH
• degenerace retiny * terapie   vrozené   MeSH
• fotoreceptory obratlovců patologie   MeSH
• fytoterapie * MeSH
• genetická terapie * metody   MeSH
• klinické zkoušky, fáze I jako téma MeSH
• klinické zkoušky, fáze II jako téma MeSH
• lidé MeSH
• mutace MeSH
• pigmentový epitel oční patologie   MeSH
• vitamin A terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Breast cancer is the most common cancer in women in the world. The role of germline genetic variability in ATP-binding cassette (ABC) transporters in cancer chemoresistance and prognosis still needs to be elucidated. We used next-generation sequencing to assess associations of germline variants in coding and regulatory sequences of all human ABC genes with response of the patients to the neoadjuvant cytotoxic chemotherapy and disease-free survival (n = 105). A total of 43 prioritized variants associating with response or survival in the above testing phase were then analyzed by allelic discrimination in the large validation set (n = 802). Variants in ABCA4, ABCA9, ABCA12, ABCB5, ABCC5, ABCC8, ABCC11, and ABCD4 associated with response and variants in ABCA7, ABCA13, ABCC4, and ABCG8 with survival of the patients. No association passed a false discovery rate test, however, the rs17822931 (Gly180Arg) in ABCC11, associating with response, and the synonymous rs17548783 in ABCA13 (survival) have a strong support in the literature and are, thus, interesting for further research. Although replicated associations have not reached robust statistical significance, the role of ABC transporters in breast cancer should not be ruled out. Future research and careful validation of findings will be essential for assessment of genetic variation which was not in the focus of this study, e.g., non-coding sequences, copy numbers, and structural variations together with somatic mutations.

• MeSH
• ABC transportéry genetika   MeSH
• alely MeSH
• frekvence genu MeSH
• genetická variace * MeSH
• genotyp MeSH
• jednonukleotidový polymorfismus MeSH
• Kaplanův-Meierův odhad MeSH
• lidé MeSH
• lokus kvantitativního znaku MeSH
• nádorové biomarkery * MeSH
• nádory prsu diagnóza   genetika   mortalita   terapie   MeSH
• neoadjuvantní terapie MeSH
• prognóza MeSH
• protokoly antitumorózní kombinované chemoterapie terapeutické užití   MeSH
• výsledek terapie MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

• MeSH
• ABC transportéry genetika   MeSH
• fenotyp MeSH
• kadherinové proteiny MeSH
• lidé MeSH
• makulární degenerace * genetika   MeSH
• mutace MeSH
• oční proteiny MeSH
• penetrance MeSH
• proteiny nervové tkáně genetika   MeSH
• retina MeSH
• rodokmen MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH